Soybean cultivar 8616350112

ABSTRACT

A soybean cultivar designated 8616350112 is disclosed. Embodiments of the invention include the seeds of soybean 8616350112, the plants of soybean 8616350112, to plant parts of soybean 8616350112, and methods for producing a soybean plant produced by crossing soybean 8616350112 with itself or with another soybean variety. Embodiments of the invention include methods for producing a soybean plant containing in its genetic material one or more genes or transgenes and the transgenic soybean plants and plant parts produced by those methods. Embodiments of the invention also relate to soybean cultivars, breeding cultivars, plant parts, and cells derived from soybean 8616350112, methods for producing other soybean cultivars, lines or plant parts derived from soybean 8616350112, and the soybean plants, varieties, and their parts derived from use of those methods. Embodiments of the invention further include hybrid soybean seeds, plants, and plant parts produced by crossing 8616350112 with another soybean cultivar.

FIELD

The present disclosure relates to a soybean cultivar designated8616350112, progeny derived therefrom, methods for using soybeancultivar 8616350112 in breeding, and the like.

BACKGROUND

All publications cited in this application are herein incorporated byreference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, and better agronomic quality.

Soybean, Glycine max (L.) Merr., is an important and valuable fieldcrop. Thus, a continuing goal of soybean plant breeders is to developstable, high yielding soybean cultivars that are agronomically sound.The reasons for this goal are to maximize the amount of grain producedon the land used and to supply food for both animals and humans. Toaccomplish this goal, the soybean breeder must select and developsoybean plants that have traits that result in superior cultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY

It is to be understood that the present invention includes a variety ofdifferent versions or embodiments, and this Summary is not meant to belimiting or all-inclusive. This Summary provides some generaldescriptions of some of the embodiments, but may also include some morespecific descriptions of other embodiments.

An embodiment of the present invention provides a soybean cultivardesignated 8616350112. Another embodiment of the present inventionrelates to the seeds of soybean cultivar 8616350112, to the plants ofsoybean cultivar 8616350112 and to methods for producing a soybean plantproduced by crossing soybean cultivar 8616350112 with itself or anothersoybean cultivar, and the creation of variants by mutagenesis ortransformation of soybean cultivar 8616350112.

Any such methods using the soybean cultivar 8616350112 are a furtherembodiment of the present invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing soybean cultivar 8616350112 as at least one parent are within thescope of this invention. Advantageously, soybean cultivar 8616350112could be used in crosses with other, different soybean plants to producefirst generation (F₁) soybean hybrid seeds and plants with superiorcharacteristics.

Another embodiment of the present invention provides for single ormultiple gene converted plants of soybean cultivar 8616350112. Thetransferred gene(s) may be a dominant or recessive allele. Thetransferred gene(s) may confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced nutritional quality, modifiedfatty acid metabolism, modified carbohydrate metabolism, modified seedyield, modified oil percent, modified protein percent, modified lodgingresistance, modified shattering, modified iron-deficiency chlorosis, andindustrial usage. The gene may be a naturally occurring soybean gene ora transgene introduced through genetic engineering techniques.

Another embodiment of the present invention provides for regenerablecells for use in tissue culture of soybean cultivar 8616350112. Thetissue culture may be capable of regenerating plants having all thephysiological and morphological characteristics of the foregoing soybeanplant, and of regenerating plants having substantially the same genotypeas the foregoing soybean plant. The regenerable cells in such tissuecultures may be embryos, protoplasts, meristematic cells, callus,pollen, leaves, ovules, anthers, cotyledons, hypocotyl, pistils, roots,root tips, flowers, seeds, petiole, pods, or stems. Still a furtherembodiment of the present invention provides for soybean plantsregenerated from the tissue cultures of soybean cultivar 8616350112.

As used herein, “at least one,” “one or more,” and “and/or” areopen-ended expressions that are both conjunctive and disjunctive inoperation. For example, each of the expressions “at least one of A, Band C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “oneor more of A, B, or C” and “A, B, and/or C” means A alone, B alone, Calone, A and B together, A and C together, B and C together, or A, B andC together.

As used herein, “sometime” means at some indefinite or indeterminatepoint of time. So for example, as used herein, “sometime after” meansfollowing, whether immediately following or at some indefinite orindeterminate point of time following the prior act.

Various embodiments of the present invention are set forth in theDetailed Description as provided herein and as embodied by the claims.It should be understood, however, that this Summary does not contain allof the aspects and embodiments of the present invention, is not meant tobe limiting or restrictive in any manner, and that the invention(s) asdisclosed herein is/are understood by those of ordinary skill in the artto encompass obvious improvements and modifications thereto.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Embryo. The embryo is the small plant contained within a mature seed.

F₃. The “F₃” symbol denotes a generation resulting from the selfing ofthe F₂ generation along with selection for type and rogueing ofoff-types. The “F” number is a term commonly used in genetics, anddesignates the number of the filial generation. The “F₃” generationdenotes the offspring resulting from the selfing or self mating ofmembers of the generation having the next lower “F” number, that is, the“F₂” generation.

Gene. Gene refers to a segment of nucleic acid. A gene can be introducedinto a genome of a species, whether from a different species or from thesame species, using transformation or various breeding methods.

Hilum. Hilum refers to the scar left on the seed that marks the placewhere the seed was attached to the pod prior to the seed beingharvested.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Iron Deficiency Chlorosis. Iron deficiency chlorosis (IDC) is ayellowing of the leaves caused by a lack of iron in the soybean plant.Iron is essential in the formation of chlorophyll, which gives plantstheir green color. In high pH soils iron becomes insoluble and cannot beabsorbed by plant roots. Soybean cultivars differ in their geneticability to utilize the available iron. A score of 9 means no stunting ofthe plants or yellowing of the leaves and a score of 1 indicates theplants are dead or dying caused by iron deficiency, a score of 5 meansplants have intermediate health with some leaf yellowing.

Linoleic Acid Percent. Linoleic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

Locus. A locus confers one or more traits such as, for example, malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism, and modified proteinmetabolism. The trait may be, for example, conferred by a naturallyoccurring gene introduced into the genome of the variety bybackcrossing, a natural or induced mutation, or a transgene introducedthrough genetic transformation techniques. A locus may comprise one ormore alleles integrated at a single chromosomal location.

Lodging Resistance. Lodging resistance refers to the relative presenceof the plant laying on or toward the ground and is on a 10 to 50 scoringbasis. A lodging score of 50 would indicate the plant is basicallylaying on the ground. A score of 10 indicates that most or all theplants in a row are standing prostrate.

Maturity Date. Plants are considered mature when 95% of the pods havereached their mature color. The number of days are calculated eitherfrom August 31 or from the planting date.

Maturity Group. Maturity group refers to an agreed-on industry divisionof groups of varieties based on zones in which they are adapted,primarily according to day length or latitude. They consist of very longday length varieties (Groups 000, 00, 0), and extend to very short daylength varieties (Groups VII, VIII, IX, X).

Oil or Oil Percent. Soybean seeds contain a considerable amount of oil.Oil is measured by NIR spectrophotometry and is reported as a percentagebasis.

Oleic Acid Percent. Oleic acid is one of the five most abundant fattyacids in soybean seeds and is measured by gas chromatography and isreported as a percent of the total oil content.

Palmitic Acid Percent. Palmitic acid is one of the five most abundantfatty acids in soybean seeds and is measured by gas chromatography andis reported as a percent of the total oil content.

Phytophthora Tolerance. Tolerance to Phytophthora root rot is rated on ascale of 1 to 9, with a score of 9 being the best or highest toleranceranging down to a score of 1 which indicates the plants have notolerance to Phytophthora.

Plant. Plant includes reference to an immature or mature whole plant,including a plant from which seed, grain, or anthers have been removed.Seed or embryo that will produce the plant is also considered to be theplant.

Plant Height. Plant height is taken from the top of the soil to the topnode of the plant and is measured in inches.

Plant Parts. Plant parts (or a soybean plant, or a part thereof)includes but is not limited to protoplasts, cells, leaves, stems, roots,root tips, anthers, pistils, seed, grain, embryo, pollen, ovules,cotyledon, hypocotyl, pod, flower, shoot, tissue, petiole, cells,meristematic cells, and the like.

Pod. Pod refers to the fruit of a soybean plant. It consists of the hullor shell (pericarp) and the soybean seeds.

Progeny. Progeny includes an F₁ soybean plant produced from the cross oftwo soybean plants where at least one plant includes soybean cultivar8616350112 and progeny further includes, but is not limited to,subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, and F₁₀ generational crosseswith the recurrent parental line.

Protein Percent. Soybean seeds contain a considerable amount of protein.Protein is generally measured by NIR spectrophotometry and is reportedon an as is percentage basis.

Pubescence. Pubescence refers to a covering of very fine hairs closelyarranged on the leaves, stems, and pods of the soybean plant.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Relative Maturity (RM). The term relative maturity is a numerical valuethat is assigned to a soybean variety based on comparisons with thematurity values of other varieties. The number preceding the decimalpoint in the RM refers to the maturity group. The number following thedecimal point refers to the relative earliness or lateness within eachmaturity group. For example, a 3.0 is an early group III variety, whilea 3.9 is a late group III variety.

Seed Yield (Bushels/Acre). The yield in bushels/acre is the actual yieldof the grain at harvest.

Seeds Per Pound. Soybean seeds vary in seed size; therefore, the numberof seeds required to make up one pound also varies. The number of seedsper pound affect the pounds of seed required to plant a given area andcan also impact end uses.

Shattering. Shattering refers to the amount of pod dehiscence prior toharvest. Pod dehiscence involves seeds falling from the pods to thesoil. This is a visual score from 1 to 9 comparing all genotypes withina given test. A score of 9 means pods have not opened and no seeds havefallen out. A score of 5 indicates approximately 50% of the pods haveopened, with seeds falling to the ground, and a score of 1 indicates100% of the pods are opened.

Single Gene Converted (Conversion). Single gene converted (conversion)plants refers to plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique or via genetic engineering.

Sulfonylurea Reaction. Sulfonylurea reaction refers to a plant'stolerance, resistance or susceptibility to sulfonylurea herbicides andrefers to a plant which contains the ALS gene, which confers resistanceto some of the sulfonylurea herbicides.

DETAILED DESCRIPTION

Soybean cultivar 8616350112 is a mid-group III maturity variety.Additionally, soybean cultivar 8616350112 is resistant to Soybean CystNematode Race 3, and contains the Rps1c which confers resistance toPhytophthora root rot.

Some of the selection criteria used for various generations include:seed yield, lodging resistance, emergence, disease tolerance, maturity,late season plant intactness, plant height, and shattering resistance.

Soybean cultivar 8616350112 has shown uniformity and stability, asdescribed in the following variety description information. Soybeancultivar 8616350112 has been self-pollinated a sufficient number ofgenerations with careful attention to uniformity of plant type and hasbeen increased with continued observation for uniformity.

Soybean cultivar 8616350112 has the following morphologic and othercharacteristics based primarily on data collected at the followinglocations: Earlham, Iowa; Lenox, Iowa; Newburg, Iowa; Walcott, Iowa;Winterset, Iowa; Chestnut, Ill.; Gilman, Ill.; Mendota, Ill.; Monmouth,Ill.; INPL, Kahoka, Mo.; Marysville, Mo.; Stromsburg, NB; Archbold,Ohio; Celina, Ohio; and Greenville, Ohio.

TABLE 1 VARIETY DESCRIPTION INFORMATION Seed Coat Color (Mature Seed):Clear Seed Coat Luster (Mature Hand Dull Shelled Seed): Color (MatureSeed): Yellow Leaflet Shape: Ovate Growth Habit: Indeterminate FlowerColor: White Hilum Color (Mature Seed): Black Plant Pubescence Color:Tawny Pod Wall Color: Brown Maturity Group: III Relative Maturity: 3.6Lodging Resistance: 18 Plant Height (cm): 103.1 Percent Protein: 43.7%Percent Oil: 17.9% Physiological Responses Resistant to Soybean Cyst(known resistances/susceptibility): Nematode Race 3, and contains theRps1c which confers resistance to Phytophthora root rot.

In Table 2, the yield of soybean cultivar 8616350112 is compared withthe yield of soybean cultivars P93Y21, AG3332, and P93M11 during summer2012 in the United States in side-by-side trials. Column one shows thesoybean cultivar designations, column two shows the number of locations,column three shows the number of observations, and column four shows theyield in bushels per acre.

TABLE 2 Soybean Cultivar # of Locations # of Observations Yield8616350112 16 16 48.2 P93Y21 16 16 51.0 8616350112 16 16 48.2 AG3332 1616 50.0 8616350112 16 16 48.2 P93M11 16 16 50.0

In Table 3, various characteristics of soybean cultivar 8616350112 arecompared with soybean cultivars AG3332, P92M61, and P93M11.

TABLE 3 Soybean Cultivar Characteristic 8616350112 AG3332 P92M31 P93M11Flower color White Purple Purple Pubescence color Tawny Tawny Gray LightTawny Hilum color Black Black Buff Black Pod color Tan Brown Brown TanSoybean Cyst Resistant Resistant Susceptible Nematode ReactionBreeding with Soybean Cultivar 8616350112

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination and the numberof hybrid offspring from each successful cross.

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of soybean plant breeding is to develop new and superiorsoybean cultivars and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selection, selfing and mutations. Therefore, a breeder will neverdevelop the same line, or even very similar lines, having the samesoybean traits from the exact same parents.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under different geographical,climatic and soil conditions and further selections are then made duringand at the end of the growing season. The cultivars that are developedare unpredictable because the breeder's selection occurs in environmentswith no control at the DNA level, and with millions of differentpossible genetic combinations being generated. A breeder of ordinaryskill in the art cannot predict the final resulting lines he develops,except possibly in a very gross and general fashion. The same breedercannot produce the same cultivar twice by using the same originalparents and the same selection techniques. This unpredictability resultsin the expenditure of large amounts of research monies to developsuperior new soybean cultivars.

The development of new soybean cultivars requires the development andselection of soybean varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as pod color, flower color, pubescence color or herbicideresistance which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Breeding programs combine desirable traits from two or more cultivars orvarious broad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. Pedigreebreeding is used commonly for the improvement of self-pollinating crops.Two parents that possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s. Selection of the best individuals may begin in the F₂ population;then, beginning in the F₃, the best individuals in the best families areselected. Replicated testing of families can begin in the F₄ generationto improve the effectiveness of selection for traits with lowheritability. At an advanced stage of inbreeding (i.e., F₆ and F₇), thebest lines or mixtures of phenotypically similar lines are tested forpotential release as new cultivars.

Using Soybean Cultivar 8616350112 to Develop Other Soybean Varieties

Soybean varieties such as soybean cultivar 8616350112 are typicallydeveloped for use in seed and grain production. However, soybeanvarieties such as soybean cultivar 8616350112 also provide a source ofbreeding material that may be used to develop new soybean varieties.Plant breeding techniques known in the art and used in a soybean plantbreeding program include, but are not limited to, recurrent selection,mass selection, bulk selection, mass selection, backcrossing, pedigreebreeding, open pollination breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection,making double haploids, and transformation. Often combinations of thesetechniques are used. The development of soybean varieties in a plantbreeding program requires, in general, the development and evaluation ofhomozygous varieties. There are many analytical methods available toevaluate a new variety. The oldest and most traditional method ofanalysis is the observation of phenotypic traits, but genotypic analysismay also be used.

Additional Breeding Methods

One embodiment of the present invention is directed to methods forproducing a soybean plant by crossing a first parent soybean plant witha second parent soybean plant, wherein the first or second soybean plantis the soybean plant from soybean cultivar 8616350112. Further, bothfirst and second parent soybean plants may be from soybean cultivar8616350112. Therefore, any methods using soybean cultivar 8616350112 arepart of this invention: selfing, backcrosses, hybrid breeding, andcrosses to populations. Any plants produced using soybean cultivar8616350112 as at least one parent are also within the scope of thisinvention. Any such methods using soybean variety 8616350112 are part ofthis invention: selfing, ribbing, backcrosses, mass selection, pedigreebreeding, bulk selection, hybrid production, crosses to populations, andthe like. These methods are well known in the art and some of the morecommonly used breeding methods are described herein. Descriptions ofbreeding methods can be found in one of several reference books (e.g.,Allard, Principles of Plant Breeding (1960); Simmonds, Principles ofCrop Improvement (1979); Sneep, et al. (1979); Fehr, “Breeding Methodsfor Cultivar Development,” Chapter 7, Soybean Improvement, Productionand Uses, 2^(nd) ed., Wilcox editor (1987)).

The following describes breeding methods that may be used with soybeancultivar 8616350112 in the development of further soybean plants. Onesuch embodiment is a method for developing a cultivar 8616350112 progenysoybean plant in a soybean plant breeding program comprising: obtainingthe soybean plant, or a part thereof, of cultivar 8616350112, utilizingsaid plant, or plant part, as a source of breeding material, andselecting a soybean cultivar 8616350112 progeny plant with molecularmarkers in common with cultivar 8616350112 and/or with morphologicaland/or physiological characteristics selected from the characteristicslisted in Tables 1 and/or 2 and/or 3 and/or 4. Breeding steps that maybe used in the soybean plant breeding program include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example, SSR markers), and the making ofdouble haploids may be utilized.

Another method involves producing a population of soybean cultivar8616350112 progeny soybean plants, comprising crossing cultivar8616350112 with another soybean plant, thereby producing a population ofsoybean plants which, on average, derive 50% of their alleles fromsoybean cultivar 8616350112. A plant of this population may be selectedand repeatedly selfed or sibbed with a soybean cultivar resulting fromthese successive filial generations. One embodiment of this invention isthe soybean cultivar produced by this method and that has obtained atleast 50% of its alleles from soybean cultivar 8616350112.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see, Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus, the invention includes soybeancultivar 8616350112 progeny soybean plants comprising a combination ofat least two cultivar 8616350112 traits selected from the groupconsisting of those listed in Tables 1 and/or 2 and/or 3 and/or 4 orsoybean cultivar 8616350112 combination of traits listed in the Summary,so that said progeny soybean plant is not significantly different forsaid traits than soybean cultivar 8616350112 as determined at the 5%significance level when grown in the same environmental conditions.Using techniques described herein, molecular markers may be used toidentify said progeny plant as a soybean cultivar 8616350112 progenyplant. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions. Once such avariety is developed, its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease resistance, pest resistance, and plantperformance in extreme environmental conditions.

Progeny of soybean cultivar 8616350112 may also be characterized throughtheir filial relationship with soybean cultivar 8616350112, as forexample, being within a certain number of breeding crosses of soybeancultivar 8616350112. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between soybean cultivar 8616350112 and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4, or 5 breeding crosses of soybean cultivar 8616350112.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which soybean plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,pods, leaves, roots, root tips, anthers, cotyledons, hypocotyls,meristematic cells, stems, pistils, petiole, and the like.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such assoybean cultivar 8616350112 and another soybean variety having one ormore desirable characteristics that is lacking or which complementssoybean cultivar 8616350112. If the two original parents do not provideall the desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations, the heterozygous condition gives way to homogeneousvarieties as a result of self-pollination and selection. Typically inthe pedigree method of breeding, five or more successive filialgenerations of selfing and selection is practiced: F₁ to F₂; F₂ to F₃;F₃ to F₄; F₄ to F₅; etc. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped variety. Preferably, the developed variety compriseshomozygous alleles at about 95% or more of its loci.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, asoybean variety may be crossed with another variety to produce a firstgeneration progeny plant. The first generation progeny plant may then bebackcrossed to one of its parent varieties to create a BC₁ or BC₂.Progeny are selfed and selected so that the newly developed variety hasmany of the attributes of the recurrent parent and yet several of thedesired attributes of the nonrecurrent parent. This approach leveragesthe value and strengths of the recurrent parent for use in new soybeanvarieties.

Therefore, an embodiment of this invention is a method of making abackcross conversion of soybean variety 8616350112, comprising the stepsof crossing a plant of soybean variety 8616350112 with a donor plantcomprising a desired trait, selecting an F₁ progeny plant comprising thedesired trait, and backcrossing the selected F₁ progeny plant to a plantof soybean variety 8616350112. This method may further comprise the stepof obtaining a molecular marker profile of soybean variety 8616350112and using the molecular marker profile to select for a progeny plantwith the desired trait and the molecular marker profile of soybeancultivar 8616350112. In one embodiment, the desired trait is a mutantgene, gene, or transgene present in the donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Soybean cultivar 8616350112 is suitablefor use in a recurrent selection program. The method entails individualplants cross pollinating with each other to form progeny. The progenyare grown and the superior progeny selected by any number of selectionmethods, which include individual plant, half-sib progeny, full-sibprogeny, and selfed progeny. The selected progeny are cross pollinatedwith each other to form progeny for another population. This populationis planted and again superior plants are selected to cross pollinatewith each other. Recurrent selection is a cyclical process and thereforecan be repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtainnew varieties for commercial or breeding use, including the productionof a synthetic cultivar. A synthetic cultivar is the resultant progenyformed by the intercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Single-Seed Descent

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

Multiple-Seed Procedure

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation in each generation of inbreeding. Enough seeds are harvestedto make up for those plants that did not germinate or produce seed.

Mutation Breeding

Mutation breeding is another method of introducing new traits intosoybean variety 8616350112. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil)), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in Fehr,“Principles of Cultivar Development,” Macmillan Publishing Company(1993). In addition, mutations created in other soybean plants may beused to produce a backcross conversion of soybean cultivar 8616350112that comprises such mutation.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

Single-Gene Conversions

When the term “soybean plant” is used in the context of an embodiment ofthe present invention, this also includes any single gene conversions ofthat variety. The term single gene converted plant as used herein refersto those soybean plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique. Backcrossing methods can be used withone embodiment of the present invention to improve or introduce acharacteristic into the variety. The term “backcrossing” as used hereinrefers to the repeated crossing of a hybrid progeny back to therecurrent parent, i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, or moretimes to the recurrent parent. The parental soybean plant thatcontributes the gene for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental soybean plant to which the geneor genes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper (1994); Fehr, Principles of CultivarDevelopment, pp. 261-286 (1987)). In a typical backcross protocol, theoriginal variety of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the single gene of interestto be transferred. The resulting progeny from this cross are thencrossed again to the recurrent parent and the process is repeated untila soybean plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic. Examples of these traits include, but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234, and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

Introduction of a New Trait or Locus into Soybean Cultivar 8616350112

Variety 8616350112 represents a new variety into which a new locus ortrait may be introgressed. Direct transformation and backcrossingrepresent two important methods that can be used to accomplish such anintrogression. The term backcross conversion and single locus conversionare used interchangeably to designate the product of a backcrossingprogram.

Backcross Conversions of Soybean Cultivar 8616350112

A backcross conversion of soybean cultivar 8616350112 occurs when DNAsequences are introduced through backcrossing (Hallauer, et al., “CornBreeding,” Corn and Corn Improvements, No. 18, pp. 463-481 (1988)), withsoybean cultivar 8616350112 utilized as the recurrent parent. Bothnaturally occurring and transgenic DNA sequences may be introducedthrough backcrossing techniques. A backcross conversion may produce aplant with a trait or locus conversion in at least two or morebackcrosses, including at least 2 crosses, at least 3 crosses, at least4 crosses, at least 5 crosses, and the like. Molecular marker assistedbreeding or selection may be utilized to reduce the number ofbackcrosses necessary to achieve the backcross conversion. For example,see, Openshaw, S. J., et al., Marker-assisted Selection in BackcrossBreeding, Proceedings Symposium of the Analysis of Molecular Data, CropScience Society of America, Corvallis, Oreg. (August 1994), where it isdemonstrated that a backcross conversion can be made in as few as twobackcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. (See, Hallauer, et al., Corn and Corn Improvement, Sprague andDudley, Third Ed. (1998)). Desired traits that may be transferredthrough backcross conversion include, but are not limited to, sterility(nuclear and cytoplasmic), fertility restoration, nutritionalenhancements, drought tolerance, nitrogen utilization, altered fattyacid profile, low phytate, industrial enhancements, disease resistance(bacterial, fungal, or viral), insect resistance, and herbicideresistance. In addition, an introgression site itself, such as an FRTsite, Lox site, or other site specific integration site, may be insertedby backcrossing and utilized for direct insertion of one or more genesof interest into a specific plant variety. In some embodiments of theinvention, the number of loci that may be backcrossed into soybeancultivar 8616350112 is at least 1, 2, 3, 4, or 5, and/or no more than 6,5, 4, 3, or 2. A single locus may contain several transgenes, such as atransgene for disease resistance that, in the same expression vector,also contains a transgene for herbicide resistance. The gene forherbicide resistance may be used as a selectable marker and/or as aphenotypic trait. A single locus conversion of site specific integrationsystem allows for the integration of multiple genes at the convertedloci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, Breeding Field Crops,p. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant, and easily recognized traits.

One process for adding or modifying a trait or locus in soybean variety8616350112 comprises crossing soybean cultivar 8616350112 plants grownfrom soybean cultivar 8616350112 seed with plants of another soybeanvariety that comprise the desired trait or locus, selecting F₁ progenyplants that comprise the desired trait or locus to produce selected F₁progeny plants, crossing the selected progeny plants with the soybeancultivar 8616350112 plants to produce backcross progeny plants,selecting for backcross progeny plants that have the desired trait orlocus and the morphological characteristics of soybean variety8616350112 to produce selected backcross progeny plants, andbackcrossing to soybean cultivar 8616350112 three or more times insuccession to produce selected fourth or higher backcross progeny plantsthat comprise said trait or locus. The modified soybean cultivar8616350112 may be further characterized as having the physiological andmorphological characteristics of soybean variety 8616350112 listed inTable 1 as determined at the 5% significance level when grown in thesame environmental conditions and/or may be characterized by percentsimilarity or identity to soybean cultivar 8616350112 as determined bySSR markers. The above method may be utilized with fewer backcrosses inappropriate situations, such as when the donor parent is highly relatedor markers are used in the selection step. Desired traits that may beused include those nucleic acids known in the art, some of which arelisted herein, that will affect traits through nucleic acid expressionor inhibition. Desired loci include the introgression of FRT, Lox, andother sites for site specific integration, which may also affect adesired trait if a functional nucleic acid is inserted at theintegration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny soybean seed byadding a step at the end of the process that comprises crossing soybeancultivar 8616350112 with the introgressed trait or locus with adifferent soybean plant and harvesting the resultant first generationprogeny soybean seed.

Molecular Techniques Using Soybean Cultivar 8616350112

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to “alter” (the utilization of up-regulation,down-regulation, or gene silencing) the traits of a plant in a specificmanner. Any DNA sequences, whether from a different species or from thesame species, which are introduced into the genome using transformationor various breeding methods are referred to herein collectively as“transgenes.” In some embodiments of the invention, a transgenic variantof soybean cultivar 8616350112 may contain at least one transgene butcould contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and/or no morethan 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Over the lastfifteen to twenty years several methods for producing transgenic plantshave been developed, and another embodiment of the present inventionalso relates to transgenic variants of the claimed soybean variety8616350112.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthineand others can also be used for antisense, dsRNA and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the invention may beproduced by any means, including genomic preparations, cDNApreparations, in-vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

One embodiment of the invention is a process for producing soybeanvariety 8616350112 further comprising a desired trait, said processcomprising introducing a transgene that confers a desired trait to asoybean plant of variety 8616350112. Another embodiment is the productproduced by this process. In one embodiment the desired trait may be oneor more of herbicide resistance, insect resistance, disease resistance,decreased phytate, or modified fatty acid or carbohydrate metabolism.The specific gene may be any known in the art or listed herein,including: a polynucleotide conferring resistance to imidazolinone,dicamba, sulfonylurea, glyphosate, glufosinate, triazine, PPO-inhibitorherbicides, benzonitrile, cyclohexanedione, phenoxy proprionic acid, andL-phosphinothricin; a polynucleotide encoding a Bacillus thuringiensispolypeptide; a polynucleotide encoding phytase, FAD-2, FAD-3, galactinolsynthase, or a raffinose synthetic enzyme; or a polynucleotideconferring resistance to soybean cyst nematode, brown stem rot,Phytophthora root rot, soybean mosaic virus, or sudden death syndrome.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993), andArmstrong, “The First Decade of Maize Transformation: A Review andFuture Perspective,” Maydica, 44:101-109 (1999). In addition, expressionvectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber, et al., “Vectors for Plant Transformation,” in Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson Eds.,CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A genetic trait which has been engineered into the genome of aparticular soybean plant may then be moved into the genome of anothervariety using traditional breeding techniques that are well known in theplant breeding arts. For example, a backcrossing approach is commonlyused to move a transgene from a transformed soybean variety into analready developed soybean variety, and the resulting backcrossconversion plant would then comprise the transgene(s).

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to, genes,coding sequences, inducible, constitutive and tissue specific promoters,enhancing sequences, and signal and targeting sequences. For example,see the traits, genes, and transformation methods listed in U.S. Pat.No. 6,118,055.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs), and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing soybean cultivar 8616350112.

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, Molecular Linkage Map ofSoybean (Glycine max L. Merr.), pp. 6.131-6.138 (1993). In S. J. O'Brien(ed.), Genetic Maps: Locus Maps of Complex Genomes, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., developed a moleculargenetic linkage map that consisted of 25 linkage groups with about 365RFLP, 11 RAPD (random amplified polymorphic DNA), 3 classical markers,and 4 isozyme loci. See also, Shoemaker, R. C., 1994 RFLP Map ofSoybean, pp. 299-309; In R. L. Phillips and I. K. Vasil (ed.), DNA-basedmarkers in plants, Kluwer Academic Press Dordrecht, the Netherlands.

SSR technology is currently the most efficient and practical markertechnology. More marker loci can be routinely used, and more alleles permarker locus can be found, using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatelliteloci in soybean with as many as 26 alleles. (Diwan, N., and Cregan. P.B., Automated sizing of fluorescent-labeled simple sequence repeat (SSR)markers to assay genetic variation in Soybean, Theor. Appl. Genet.,95:220-225 (1997)). Single Nucleotide Polymorphisms may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Soybean DNA molecular marker linkage maps have been rapidly constructedand widely implemented in genetic studies. One such study is describedin Cregan, et. al, “An Integrated Genetic Linkage Map of the SoybeanGenome,” Crop Science, 39:1464-1490 (1999). Sequences and PCR conditionsof SSR Loci in Soybean, as well as the most current genetic map, may befound in Soybase on the World Wide Web.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a soybean plant for which soybean cultivar 8616350112 is aparent can be used to produce double haploid plants. Double haploids areproduced by the doubling of a set of chromosomes (1N) from aheterozygous plant to produce a completely homozygous individual. Forexample, see, Wan, et al., “Efficient Production of Doubled HaploidPlants Through Colchicine Treatment of Anther-Derived Maize Callus,”Theoretical and Applied Genetics, 77:889-892 (1989) and U.S. Pat. No.7,135,615. This can be advantageous because the process omits thegenerations of selfing needed to obtain a homozygous plant from aheterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, Am. Nat., 93:381-382 (1959); Sharkar and Coe, Genetics,54:453-464 (1966); KEMS (Deimling, Roeber, and Geiger, Vortr.Pflanzenzuchtg, 38:203-224 (1997); or KMS and ZMS (Chalyk, Bylich &Chebotar, MNL, 68:47 (1994); Chalyk & Chebotar, Plant Breeding,119:363-364 (2000)); and indeterminate gametophyte (ig) mutation(Kermicle, Science, 166:1422-1424 (1969)). The disclosures of which areincorporated herein by reference.

Methods for obtaining haploid plants are also disclosed in Kobayashi,M., et al., Journ. of Heredity, 71(1):9-14 (1980); Pollacsek, M.,Agronomie (Paris) 12(3):247-251 (1992); Cho-Un-Haing, et al., Journ. ofPlant Biol., 39(3):185-188 (1996); Verdoodt, L., et al., 96(2):294-300(February 1998); Chalyk, et al., Maize Genet Coop., Newsletter 68:47(1994).

Thus, an embodiment of this invention is a process for making asubstantially homozygous soybean cultivar 8616350112 progeny plant byproducing or obtaining a seed from the cross of soybean cultivar8616350112 and another soybean plant and applying double haploid methodsto the F₁ seed or F₁ plant or to any successive filial generation. Basedon studies in maize and currently being conducted in soybean, suchmethods would decrease the number of generations required to produce avariety with similar genetics or characteristics to soybean cultivar8616350112. See, Bernardo, R. and Kahler, A. L., Theor. Appl. Genet.,102:986-992 (2001).

In particular, a process of making seed retaining the molecular markerprofile of soybean variety 8616350112 is contemplated, such processcomprising obtaining or producing F₁ seed for which soybean variety8616350112 is a parent, inducing doubled haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of soybean variety 8616350112, and selecting progeny thatretain the molecular marker profile of soybean cultivar 8616350112.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard (1960); Simmonds (1979); Sneep, et al. (1979); Fehr(1987))

Expression Vectors for Soybean Transformation: Marker Genes

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). Expression vectors includeat least one genetic marker operably linked to a regulatory element (forexample, a promoter) that allows transformed cells containing the markerto be either recovered by negative selection, i.e., inhibiting growth ofcells that do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well-known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or an herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley, et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983).

Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford, et al.,Plant Physiol., 86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86(1987); Svab, et al., Plant Mol. Biol., 14:197 (1990); Hille, et al.,Plant Mol. Biol., 7:171 (1986)). Other selectable marker genes conferresistance to herbicides such as glyphosate, glufosinate, or bromoxynil(Comai, et al., Nature, 317:741-744 (1985); Gordon-Kamm, et al., PlantCell, 2:603-618 (1990); Stalker, et al., Science, 242:419-423 (1988)).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase (Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); Charest, et al., Plant Cell Rep.,8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells, rather than directgenetic selection of transformed cells, for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock, et al., EMBO J., 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available (Molecular Probes, Publication2908, IMAGENE GREEN, pp. 1-4 (1993); Naleway, et al., J. Cell Biol.,115:151a (1991)). However, these in-vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds, andlimitations associated with the use of luciferase genes as selectablemarkers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie, et al., Science, 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Soybean Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

A. Inducible Promoters: An inducible promoter is operably linked to agene for expression in soybean. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in soybean. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See, Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Mett, et al., Proc. Natl. Acad. Sci. USA,90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey, et al., Mol. Gen.Genetics, 227:229-237 (1991); Gatz, et al., Mol. Gen. Genetics,243:32-38 (1994)); or Tet repressor from Tn10 (Gatz, et al., Mol. Gen.Genetics, 227:229-237 (1991)). A particularly preferred induciblepromoter is a promoter that responds to an inducing agent to whichplants do not normally respond. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena,et al., Proc. Natl. Acad. Sci. USA, 88:0421 (1991)).

B. Constitutive Promoters: A constitutive promoter is operably linked toa gene for expression in soybean or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in soybean.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989);Christensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)); and maize H3 histone (Lepetit, et al.,Mol. Gen. Genetics, 231:276-285 (1992); Atanassova, et al., PlantJournal, 2 (3):291-300 (1992)). The ALS promoter, XbaI/NcoI fragment 5′to the Brassica napus ALS3 structural gene (or a nucleotide sequencesimilarity to said XbaI/NcoI fragment), represents a particularly usefulconstitutive promoter. See, U.S. Pat. No. 5,659,026.

C. Tissue-Specific or Tissue-Preferred Promoters: A tissue-specificpromoter is operably linked to a gene for expression in soybean.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in soybean. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983); Sengupta-Gopalan, et al., Proc. Natl. Acad. Sci. USA,82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson, et al., EMBO J., 4(11):2723-2729(1985); Timko, et al., Nature, 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell, et al., Mol. Gen. Genetics,217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero, et al., Mol. Gen. Genetics, 244:161-168 (1993)); or amicrospore-preferred promoter such as that from apg (Twell, et al., Sex.Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment, such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Knox, C., etal., Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., Plant Physiol.,91:124-129 (1989); Frontes, et al., Plant Cell, 3:483-496 (1991);Matsuoka, et al., Proc. Natl. Acad. Sci., 88:834 (1991); Gould, et al.,J. Cell. Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129(1991); Kalderon, et al., Cell, 39:499-509 (1984); Steifel, et al.,Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes: Transformation

With transgenic plants according to one embodiment of the presentinvention, a foreign protein can be produced in commercial quantities.Thus, techniques for the selection and propagation of transformedplants, which are well understood in the art, yield a plurality oftransgenic plants which are harvested in a conventional manner, and aforeign protein can then be extracted from a tissue of interest or fromtotal biomass. Protein extraction from plant biomass can be accomplishedby known methods which are discussed, for example, by Heney and Orr,Anal. Biochem., 114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a soybean plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see, Glick and Thompson, Methods in PlantMolecular Biology and Biotechnology, CRC Press, Inc., Boca Raton,269:284 (1993). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant.

Wang, et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome,” Science,280:1077-1082 (1998), and similar capabilities are becoming increasinglyavailable for the soybean genome. Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR, and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Likewise, by means of one embodiment of the present invention, plantscan be genetically engineered to express various phenotypes of agronomicinterest. Through the transformation of soybean, the expression of genescan be altered to enhance disease resistance, insect resistance,herbicide resistance, agronomic, grain quality, and other traits.Transformation can also be used to insert DNA sequences which control orhelp control male-sterility. DNA sequences native to soybean, as well asnon-native DNA sequences, can be transformed into soybean and used toalter levels of native or non-native proteins. Various promoters,targeting sequences, enhancing sequences, and other DNA sequences can beinserted into the genome for the purpose of altering the expression ofproteins. The interruption or suppression of the expression of a gene atthe level of transcription or translation (also known as gene silencingor gene suppression) is desirable for several aspects of geneticengineering in plants.

Many techniques for gene silencing are well-known to one of skill in theart, including, but not limited to, knock-outs (such as by insertion ofa transposable element such as Mu (Vicki Chandler, The Maize Handbook,Ch. 118 (Springer-Verlag 1994)) or other genetic elements such as a FRT,Lox, or other site specific integration sites; antisense technology(see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809 (1988) and U.S. Pat.Nos. 5,107,065, 5,453,566, and 5,759,829); co-suppression (e.g., Taylor,Plant Cell, 9:1245 (1997); Jorgensen, Trends Biotech., 8(12):340-344(1990); Flavell, PNAS USA, 91:3490-3496 (1994); Finnegan, et al.,Bio/Technology, 12:883-888 (1994); Neuhuber, et al., Mol. Gen. Genet.,244:230-241 (1994)); RNA interference (Napoli, et al., Plant Cell,2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes Dev., 13:139-141(1999); Zamore, et al., Cell, 101:25-33 (2000); Montgomery, et al., PNASUSA, 95:15502-15507 (1998)), virus-induced gene silencing (Burton, etal., Plant Cell, 12:691-705 (2000); Baulcombe, Curr. Op. Plant Bio.,2:109-113 (1999)); target-RNA-specific ribozymes (Haseloff, et al.,Nature, 334:585-591 (1988)); hairpin structures (Smith, et al., Nature,407:319-320 (2000); U.S. Pat. Nos. 6,423,885, 7,138,565, 6,753,139, and7,713,715); MicroRNA (Aukerman & Sakai, Plant Cell, 15:2730-2741(2003)); ribozymes (Steinecke, et al., EMBO J., 11:1525 (1992);Perriman, et al., Antisense Res. Dev., 3:253 (1993)); oligonucleotidemediated targeted modification (e.g., U.S. Pat. Nos. 6,528,700 and6,911,575); Zn-finger targeted molecules (e.g., U.S. Pat. Nos.7,151,201, 6,453,242, 6,785,613, 7,177,766 and 7,788,044); and othermethods or combinations of the above methods known to those of skill inthe art.

Methods for Soybean Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in-vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation,” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-mediated Transformation: One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch, et al., Science,227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci., 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber, et al., supra, Miki, et al., supra, andMoloney, et al., Plant Cell Reports, 8:238 (1989). See also, U.S. Pat.No. 5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer: Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation where DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford, et al., Part. Sci. Technol., 5:27 (1987);Sanford, J. C., Trends Biotech., 6:299 (1988); Klein, et al., Bio/Tech.,6:559-563 (1988); Sanford, J. C., Physiol Plant, 7:206 (1990); Klein, etal., Biotechnology, 10:268 (1992). See also, U.S. Pat. No. 5,015,580(Christou, et al.), issued May 14, 1991 and U.S. Pat. No. 5,322,783.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/Technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985); Christou, et al., Proc Natl. Acad. Sci. USA, 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine have also been reported. Hain, etal., Mol. Gen. Genet., 199:161 (1985) and Draper, et al., Plant CellPhysiol., 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues has also been described (D'Halluin, et al., Plant Cell,4:1495-1505 (1992); and Spencer, et al., Plant Mol. Biol., 24:51-61(1994)).

Following transformation of soybean target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues, and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety in orderto produce a new transgenic variety. Alternatively, a genetic trait thathas been engineered into a particular soybean line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple x by y cross or the process ofbackcrossing depending on the context.

Likewise, by means of one embodiment of the present invention, agronomicgenes can be expressed in transformed plants. More particularly, plantscan be genetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with one ormore cloned resistance genes to engineer plants that are resistant tospecific pathogen strains. See, for example, Jones, et al., Science,266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin, et al., Science, 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. tomato encodes aprotein kinase); Mindrinos, et al., Cell, 78:1089 (1994) (ArabidopsisRSP2 gene for resistance to Pseudomonas syringae); McDowell & Woffenden,Trends Biotechnol., 21(4):178-83 (2003); and Toyoda, et al., TransgenicRes., 11 (6):567-82 (2002).

B. A gene conferring resistance to a pest, such as soybean cystnematode. See, e.g., U.S. Pat. No. 5,994,627.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser, et al., Gene,48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding 6-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

D. A lectin. See, for example, Van Damme, et al., Plant Molec. Biol.,24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See, InternationalApplication No. PCT/US 1993/006487, which teaches the use of avidin andavidin homologues as larvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Molec. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani, etal., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor); and U.S. Pat. No.5,494,813.

G. An insect-specific hormone or pheromone, such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor); Pratt, et al.,Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., CriticalReviews in Microbiology, 30(1):33-54 (2004); Zjawiony, J. Nat. Prod.,67(2):300-310 (2004); Carlini & Grossi-de-Sa, Toxicon, 40(11):1515-1539(2002); Ussuf, et al., Curr Sci., 80(7):847-853 (2001); Vasconcelos &Oliveira, Toxicon, 44(4):385-403 (2004). See also, U.S. Pat. No.5,266,317 which discloses genes encoding insect-specific, paralyticneurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see, Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See, U.S. Pat.No. 5,955,653 which discloses the nucleotide sequence of a callase gene.DNA molecules which contain chitinase-encoding sequences can beobtained, for example, from the ATCC under Accession Nos. 39637 and67152. See also, Kramer, et al., Insect Biochem. Molec. Biol., 23:691(1993), who teach the nucleotide sequence of a cDNA encoding tobaccohornworm chitinase, and Kawalleck, et al., Plant Molec. Biol., 21:673(1993), who provide the nucleotide sequence of the parsley ubi4-2polyubiquitin gene, U.S. Pat. Nos. 7,145,060, 7,087,810, and 6,563,020.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Molec. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See, U.S. Pat. No. 5,580,852, whichdiscloses peptide derivatives of tachyplesin which inhibit fungal plantpathogens, and U.S. Pat. No. 5,607,914 which teaches syntheticantimicrobial peptides that confer disease resistance.

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci, 89:43 (1993),of heterologous expression of a cecropin-13 lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,and tobacco mosaic virus.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

Q. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See, Lamb, et al., Bio/Technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J., 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/Technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

T. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S., Current Biology, 5(2)(1995); Pieterse & Van Loon, Curr. Opin. Plant Bio., 7(4):456-64 (2004);and Somssich, Cell, 113(7):815-6 (2003).

U. Antifungal genes. See, Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs, et al., Planta, 183:258-264 (1991); andBushnell, et al., Can. J of Plant Path., 20(2):137-149 (1998). See also,U.S. Pat. No. 6,875,907.

V. Detoxification genes, such as for fumonisin, beauvericin,moniliformin, and zearalenone and their structurally-relatedderivatives. See, U.S. Pat. No. 5,792,931.

W. Cystatin and cysteine proteinase inhibitors. See, U.S. Pat. No.7,205,453.

X. Defensin genes. See, U.S. Pat. Nos. 6,911,577, 7,855,327, 7855,328,7,897,847, 7,910,806, 7,919,686, and 8,026,415.

Y. Genes conferring resistance to nematodes, and in particular soybeancyst nematodes. See, U.S. Pat. Nos. 5,994,627 and 6,294,712; Urwin, etal., Planta, 204:472-479 (1998); Williamson, Curr Opin Plant Bio.,2(4):327-31 (1999).

Z. Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7, and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

AA. Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

Any of the above-listed disease or pest resistance genes (A-AA) can beintroduced into the claimed soybean cultivar through a variety of meansincluding, but not limited to, transformation and crossing.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988) and Mild, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), pyridinoxy or phenoxy proprionic acids,and cyclohexanediones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 which discloses the nucleotide sequenceof a form of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 which describes genes encoding EPSPS enzymes. See also, U.S.Pat. Nos. 6,566,587, 6,338,961, 6,248,876, 6,040,497, 5,804,425,5,633,435, 5,145,783, 4,971,908, 5,312,910, 5,188,642, 4,940,835,5,866,775, 6,225,114, 6,130,366, 5,310,667, 4,535,060, 4,769,061,5,633,448, 5,510,471, 6,803,501, RE 36,449, RE 37,287, and 5,491,288,which are incorporated herein by reference for this purpose. Glyphosateresistance is also imparted to plants that express a gene that encodes aglyphosate oxido-reductase enzyme, as described more fully in U.S. Pat.Nos. 5,776,760 and 5,463,175, which are incorporated herein by referencefor this purpose. In addition, glyphosate resistance can be imparted toplants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. Pat. No. 7,462,481. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCAccession No. 39256, and the nucleotide sequence of the mutant gene isdisclosed in U.S. Pat. No. 4,769,061. European Patent Appl. No. 0333033and U.S. Pat. No. 4,975,374 disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean Patent No. 0242246 to Leemans, et al. DeGreef, et al.,Bio/Technology, 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibila, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992). Protoporphyrinogen oxidase (PPO) is the target of thePPO-inhibitor class of herbicides; a PPO-inhibitor resistant PPO genewas recently identified in Amaranthus tuberculatus (Patzoldt et al.,PNAS, 103(33):12329-2334, 2006). The herbicide methyl viologen inhibitsCO₂ assimilation. Foyer et al. (Plant Physiol., 109:1047-1057, 1995)describe a plant overexpressing glutathione reductase (GR) which isresistant to methyl viologen treatment. Bromoxynil resistance byintroducing a chimeric gene containing the bxn gene (Science, 242(4877):419-23, 1988).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, Hattori, et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17 (1994)); genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)); and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619(1992)).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and 6,084,155.

Any of the above listed herbicide genes (A-E) can be introduced into theclaimed soybean cultivar through a variety of means including but notlimited to transformation and crossing.

3. Genes that Confer or Contribute to a Value-Added Trait, such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See, Knultzon, et al., Proc. Natl. Acad. Sci.USA, 89:2625 (1992).

B. Decreased phytate content: 1) Introduction of a phytase-encoding geneenhances breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see, Van Hartingsveldt, et al., Gene,127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) Up-regulation of a gene that reducesphytate content. In maize, this, for example, could be accomplished bycloning and then re-introducing DNA associated with one or more of thealleles, such as the LPA alleles, identified in maize mutantscharacterized by low levels of phytic acid, such as in Raboy, et al.,Maydica, 35:383 (1990), and/or by altering inositol kinase activity asin, for example, U.S. Pat. Nos. 7,425,442, 7,714,187, 6,197,561,6,2191,224, 6,855,869, 6,391,348, 6,197,561, and 6,291,224; U.S. Publ.Nos. 2003/000901, 2003/0009011, and 2006/272046; and International Pub.Nos. WO 98/45448, and WO 01/04147.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch, or a gene altering thioredoxin, such as NTRand/or TRX (See, U.S. Pat. No. 6,531,648, which is incorporated byreference for this purpose), and/or a gamma zein knock out or mutant,such as cs27 or TUSC27 or en27 (See, U.S. Pat. Nos. 6,858,778, 7,741,533and U.S. Publ. No. 2005/0160488, which are incorporated by reference forthis purpose). See, Shiroza, et al., J. Bacteriol., 170:810 (1988)(nucleotide sequence of Streptococcus mutans fructosyltransferase gene);Steinmetz, et al., Mol. Gen. Genet., 200:220 (1985) (nucleotide sequenceof Bacillus subtilis levansucrase gene); Pen, et al., Bio/Technology,10:292 (1992) (production of transgenic plants that express Bacilluslichenifonnis α-amylase); Elliot, et al., Plant Molec. Biol., 21:515(1993) (nucleotide sequences of tomato invertase genes); Søgaard, etal., J. Biol. Chem., 268:22480-22484 (1993) (site-directed mutagenesisof barley α-amylase gene); Fisher, et al., Plant Physiol., 102:1045(1993) (maize endosperm starch branching enzyme II); International Pub.No. WO 99/10498 (improved digestibility and/or starch extraction throughmodification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref 1, HCHL,C4H); U.S. Pat. No. 6,232,529 (method of producing high oil seed bymodification of starch levels (AGP)). The fatty acid modification genesmentioned above may also be used to affect starch content and/orcomposition through the interrelationship of the starch and oilpathways.

D. Elevated oleic acid via FAD-2 gene modification and/or decreasedlinolenic acid via FAD-3 gene modification. See, U.S. Pat. Nos.5,952,544, 6,063,947, and 6,323,392. Linolenic acid is one of the fivemost abundant fatty acids in soybean seeds. The low oxidative stabilityof linolenic acid is one reason that soybean oil undergoes partialhydrogenation. When partially hydrogenated, all unsaturated fatty acidsform trans fats. Soybeans are the largest source of edible-oils in theU.S. and 40% of soybean oil production is partially hydrogenated. Theconsumption of trans fats increases the risk of heart disease.Regulations banning trans fats have encouraged the development of lowlinolenic soybeans. Soybeans containing low linolenic acid percentagescreate a more stable oil requiring hydrogenation less often. Thisprovides trans fat free alternatives in products such as cooking oil.

E. Altering conjugated linolenic or linoleic acid content, such as inU.S. Pat. No. 6,593,514. Altering LEC1, AGP, Dek1, Superal1, milps, andvarious Ipa genes, such as Ipa1, Ipa3, hpt, or hggt. See, for example,U.S. Pat. Nos. 7,122,658, 7,342,418, 6,232,529, 7,888,560, 6,423,886,6,197,561, 6,825,397 and 7,157,621; U.S. Publ. No. 2003/0079247;International Publ. No. WO 2003/011015; and Rivera-Madrid, R., et al.,Proc. Natl. Acad. Sci., 92:5620-5624 (1995).

F. Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. See, for example, U.S. Pat. Nos. 6,787,683,7,154,029 and International Publ. No. WO 00/68393 (involving themanipulation of antioxidant levels through alteration of a phytl prenyltransferase (ppt)); and U.S. Pat. Nos. 7,154,029 and 7,622,658 (throughalteration of a homogentisate geranyl geranyl transferase (hggt)).

G. Altered essential seed amino acids. See, for example, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds); U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds); U.S. Pat. No. 5,990,389and International Publ. No. WO 95/15392 (high lysine); U.S. Pat. No.5,850,016 (alteration of amino acid compositions in seeds); U.S. Pat.No. 5,885,802 (high methionine); U.S. Pat. No. 5,885,801 andInternational Publ. No. WO96/01905 (high threonine); U.S. Pat. Nos.6,664,445, 7,022,895, 7,368,633, and 7,439,420 (plant amino acidbiosynthetic enzymes); U.S. Pat. No. 6,459,019 and U.S. application Ser.No. 09/381,485 (increased lysine and threonine); U.S. Pat. No. 6,441,274(plant tryptophan synthase beta subunit); U.S. Pat. No. 6,346,403(methionine metabolic enzymes); U.S. Pat. No. 5,939,599 (high sulfur);U.S. Pat. No. 5,912,414 (increased methionine); U.S. Pat. No. 5,633,436(increasing sulfur amino acid content); U.S. Pat. No. 5,559,223(synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants); U.S. Pat. No. 6,194,638 (hemicellulose);U.S. Pat. No. 7,098,381 (UDPGdH); U.S. Pat. No. 6,194,638 (RGP); U.S.Pat. Nos. 6,399,859, 6,930,225, 7,179,955, 6,803,498, 5,850,016, and7,053,282 (alteration of amino acid compositions in seeds); WO 99/29882(methods for altering amino acid content of proteins); U.S. applicationSer. No. 09/297,418 (proteins with enhanced levels of essential aminoacids); WO 98/45458 (engineered seed protein having higher percentage ofessential amino acids); WO 01/79516; and U.S. Pat. Nos. 6,803,498,6,930,225, 7,307,149, 7,524,933, 7,579,443, 7,838,632, 7,851,597, and7,982,009 (maize cellulose synthases).

4. Genes that Control Male Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describes a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on,”the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac—PPT. See, U.S. Pat. No. 6,384,304.

B. Introduction of various stamen-specific promoters. See, U.S. Pat.Nos. 5,639,948 and 5,589,610.

C. Introduction of the barnase and the barstar genes. See, Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341, 6,297,426, 5,478,369,5,824,524, 5,850,014, and 6,265,640, all of which are herebyincorporated by reference.

5. Genes that Create a Site for Site Specific DNA Integration:

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.See, for example, Lyznik, et al., Site-Specific Recombination forGenetic Engineering in Plants, Plant Cell Rep, 21:925-932 (2003) andU.S. Pat. No. 6,187,994, which are hereby incorporated by reference.Other systems that may be used include the Gin recombinase of phage Mu(Maeser, et al. (1991); Vicki Chandler, The Maize Handbook, Ch. 118(Springer-Verlag 1994)); the Pin recombinase of E. coli (Enomoto, et al.(1983)); and the R/RS system of the pSRi plasmid (Araki, et al. (1992)).

6. Genes that Affect Abiotic Stress Resistance:

Genes that affect abiotic stress resistance (including but not limitedto flowering, pod and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see U.S. Pat. No. 6,653,535 where water use efficiency is alteredthrough alteration of malate; U.S. Pat. Nos. 5,892,009, 5,965,705,5,929,305, 5,891,859, 6,417,428, 6,664,446, 6,706,866, 6,717,034,6,801,104, 6,946,586, 7,238,860, 7,635,800, 7,135,616, 7,193,129, and7,601,893; and International Publ. Nos. WO 2001/026459, WO 2001/035725,WO 2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO2002/015675, and WO 2002/077185, describing genes, including CBF genesand transcription factors effective in mitigating the negative effectsof freezing, high salinity, and drought on plants, as well as conferringother positive effects on plant phenotype; U.S. Publ. No. 2004/0148654,where abscisic acid is altered in plants resulting in improved plantphenotype, such as increased yield and/or increased tolerance to abioticstress; U.S. Pat. Nos. 6,992,237, 6,429,003, 7,049,115, and 7,262,038,where cytokinin expression is modified resulting in plants withincreased stress tolerance, such as drought tolerance, and/or increasedyield. See also, WO 02/02776, WO 2003/052063, JP 2002281975, U.S. Pat.No. 6,084,153, WO 01/64898, and U.S. Pat. Nos. 6,177,275 and 6,107,547(enhancement of nitrogen utilization and altered nitrogenresponsiveness). For ethylene alteration, see, U.S. Publ. Nos.2004/0128719, 2003/0166197, and U.S. application Ser. No. 09/856,834.For plant transcription factors or transcriptional regulators of abioticstress, see, e.g., U.S. Publ. Nos. 2004/0098764 or 2004/0078852.

Other genes and transcription factors that affect plant growth andagronomic traits, such as yield, flowering, plant growth, and/or plantstructure, can be introduced or introgressed into plants. See forexample, U.S. Pat. Nos. 6,140,085, and 6,265,637 (CO); U.S. Pat. No.6,670,526 (ESD4); U.S. Pat. Nos. 6,573,430 and 7,157,279 (TFL); U.S.Pat. No. 6,713,663 (FT); U.S. Pat. Nos. 6,794,560, 6,307,126 (GAD; U.S.Pat. No. 7,045,682 (VRN1); U.S. Pat. Nos. 6,949,694 and 7,253,274(VRN2); U.S. Pat. No. 6,887,708 (GI); U.S. Pat. No. 7,320,158 (FRI);U.S. Pat. No. 6,307,126 (GAD; U.S. Pat. Nos. 6,762,348 and 7,268,272 (D8and Rht); and U.S. Pat. Nos. 7,345,217, 7,511,190, 7,659,446, and7,825,296 (transcription factors).

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety, ora related variety, or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) (which are also referred to asMicrosatellites), and Single Nucleotide Polymorphisms (SNPs). Forexample, see, Cregan, et al., “An Integrated Genetic Linkage Map of theSoybean Genome,” Crop Science, 39:1464-1490 (1999) and Berry, et al.,“Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles: Applications to Maize Inbred Lines and Soybean Varieties,”Genetics, 165:331-342 (2003), each of which are incorporated byreference herein in their entirety.

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forsoybean cultivar 8616350112.

Primers and PCR protocols for assaying these and other markers aredisclosed in the Soybase (sponsored by the USDA Agricultural ResearchService and Iowa State University). In addition to being used foridentification of soybean variety 8616350112, and plant parts and plantcells of soybean variety 8616350112, the genetic profile may be used toidentify a soybean plant produced through the use of soybean cultivar8616350112 or to verify a pedigree for progeny plants produced throughthe use of soybean cultivar 8616350112. The genetic marker profile isalso useful in breeding and developing backcross conversions.

One embodiment of the present invention comprises a soybean plantcharacterized by molecular and physiological data obtained from thesample of said variety deposited with the American Type CultureCollection (ATCC) or with the National Collections of Industrial, Foodand Marine Bacteria (NCIMB). Further provided by the invention is asoybean plant formed by the combination of the disclosed soybean plantor plant cell with another soybean plant or cell and comprising thehomozygous alleles of the variety. “Cell” as used herein includes aplant cell, whether isolated, in tissue culture or incorporated in aplant or plant part.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may be present(“linkage” refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent). Another advantage of this type of markeris that, through use of flanking primers, detection of SSRs can beachieved, for example, by the polymerase chain reaction (PCR), therebyeliminating the need for labor-intensive Southern hybridization. The PCRdetection is done by use of two oligonucleotide primers flanking thepolymorphic segment of repetitive DNA. Repeated cycles of heatdenaturation of the DNA followed by annealing of the primers to theircomplementary sequences at low temperatures, and extension of theannealed primers with DNA polymerase, comprise the major part of themethodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties it is preferable if all SSRprofiles are performed in the same lab.

Primers used are publicly available and may be found in the Soybase orCregan supra. See also, U.S. application Ser. No. 09/581,970 (NucleotidePolymorphisms in Soybean); U.S. Pat. No. 6,162,967 (Positional Cloningof Soybean Cyst Nematode Resistance Genes); and U.S. Pat. No. 7,288,386(Soybean Sudden Death Syndrome Resistant Soybeans and Methods ofBreeding and Identifying Resistant Plants), the disclosure of which areincorporated herein by reference.

The SSR profile of soybean plant 8616350112 can be used to identifyplants comprising soybean cultivar 8616350112 as a parent, since suchplants will comprise the same homozygous alleles as soybean cultivar8616350112. Because the soybean variety is essentially homozygous at allrelevant loci, most loci should have only one type of allele present. Incontrast, a genetic marker profile of an F₁ progeny should be the sum ofthose parents, e.g., if one parent was homozygous for allele x at aparticular locus, and the other parent homozygous for allele y at thatlocus, then the F₁ progeny will be xy (heterozygous) at that locus.Subsequent generations of progeny produced by selection and breeding areexpected to be of genotype x (homozygous), y (homozygous), or xy(heterozygous) for that locus position. When the F₁ plant is selfed orsibbed for successive filial generations, the locus should be either xor y for that position.

In addition, plants and plant parts substantially benefiting from theuse of soybean cultivar 8616350112 in their development, such as soybeancultivar 8616350112 comprising a backcross conversion, transgene, orgenetic sterility factor, may be identified by having a molecular markerprofile with a high percent identity to soybean cultivar 8616350112.Such a percent identity might be 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% identical to soybean cultivar 8616350112. Percent identity refersto the comparison of the homozygous alleles of two soybean varieties.Percent identity or percent similarity is determined by comparing astatistically significant number of the homozygous alleles of twodeveloped varieties. For example, a percent identity of 90% betweensoybean variety 1 and soybean variety 2 means that the two varietieshave the same allele at 90% of their loci.

The SSR profile of soybean cultivar 8616350112 can also be used toidentify essentially derived varieties and other progeny varietiesdeveloped from the use of soybean cultivar 8616350112, as well as cellsand other plant parts thereof. Such plants may be developed using themarkers, for example, identified in U.S. Pat. Nos. 6,162,967, and7,288,386. Progeny plants and plant parts produced using soybeancultivar 8616350112 may be identified by having a molecular markerprofile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% geneticcontribution from soybean variety, as measured by either percentidentity or percent similarity. Such progeny may be furthercharacterized as being within a pedigree distance of soybean cultivar8616350112, such as within 1, 2, 3, 4, or 5 or less cross-pollinationsto a soybean plant other than soybean cultivar 8616350112 or a plantthat has soybean cultivar 8616350112 as a progenitor. Unique molecularprofiles may be identified with other molecular tools such as SNPs andRFLPs.

While determining the SSR genetic marker profile of the plants describedsupra, several unique SSR profiles may also be identified which did notappear in either parent of such plant. Such unique SSR profiles mayarise during the breeding process from recombination or mutation. Acombination of several unique alleles provides a means of identifying aplant variety, an F₁ progeny produced from such variety, and progenyproduced from such variety.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well-known and widely published. Forexample, reference may be had to Komatsuda, T., et al., Crop Sci.,31:333-337 (1991); Stephens, P. A., et al., Theor. Appl. Genet.,82:633-635 (1991); Komatsuda, T., et al., Plant Cell, Tissue and OrganCulture, 28:103-113 (1992); Dhir, S., et al., Plant Cell Reports,11:285-289 (1992); Pandey, P., et al., Japan J. Breed., 42:1-5 (1992);and Shetty, K., et al., Plant Science, 81:245-251 (1992); as well asU.S. Pat. No. 5,024,944 and U.S. Pat. No. 5,008,200. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce soybean plants having the physiological andmorphological characteristics of soybean cultivar 8616350112.

Regeneration refers to the development of a plant from tissue culture.The term “tissue culture” indicates a composition comprising isolatedcells of the same or a different type or a collection of such cellsorganized into parts of a plant. Exemplary types of tissue cultures areprotoplasts, calli, plant clumps, and plant cells that can generatetissue culture that are intact in plants or parts of plants, such asembryos, pollen, flowers, seeds, pods, petioles, leaves, stems, roots,root tips, anthers, pistils, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and 5,977,445describe certain techniques, the disclosures of which are incorporatedherein by reference.

Industrial Uses

The seed of soybean cultivar 8616350112, the plant produced from theseed, the hybrid soybean plant produced from the crossing of the varietywith any other soybean plant, hybrid seed, and various parts of thehybrid soybean plant can be utilized for human food, livestock feed, andas a raw material in industry. The soybean seeds produced by soybeancultivar 8616350112 can be crushed, or a component of the soybean seedscan be extracted, in order to comprise a commodity plant product, suchas protein concentrate, protein isolate, soybean hulls, meal, flour, oroil for a food or feed product.

The soybean is the world's leading source of vegetable oil and proteinmeal. The oil extracted from soybeans is used for cooking oil,margarine, and salad dressings. Soybean oil is composed of saturated,monounsaturated, and polyunsaturated fatty acids. It has a typicalcomposition of 11% palmitic, 4% stearic, 25% oleic, 50% linoleic, and 9%linolenic fatty acid content (“Economic Implications of Modified SoybeanTraits Summary Report,” Iowa Soybean Promotion Board and AmericanSoybean Association Special Report 92S (May 1990)). Changes in fattyacid composition for improved oxidative stability and nutrition areconstantly sought after. Industrial uses of soybean oil, which issubjected to further processing, include ingredients for paints,plastics, fibers, detergents, cosmetics, lubricants, and biodiesel fuel.Soybean oil may be split, inter-esterified, sulfurized, epoxidized,polymerized, ethoxylated, or cleaved. Designing and producing soybeanoil derivatives with improved functionality and improved oliochemistryis a rapidly growing field. The typical mixture of triglycerides isusually split and separated into pure fatty acids, which are thencombined with petroleum-derived alcohols or acids, nitrogen, sulfonates,chlorine, or with fatty alcohols derived from fats and oils to producethe desired type of oil or fat.

Soybean cultivar 8616350112 can be used to produce soybean oil. Toproduce soybean oil, the soybeans harvested from soybean cultivar8616350112 are cracked, adjusted for moisture content, rolled intoflakes and the oil is solvent-extracted from the flakes with commercialhexane. The oil is then refined, blended for different applications, andsometimes hydrogenated. Soybean oils, both liquid and partiallyhydrogenated, are used domestically and exported, sold as “vegetableoil” or are used in a wide variety of processed foods.

Soybeans are also used as a food source for both animals and humans.Soybeans are widely used as a source of protein for poultry, swine, andcattle feed. During processing of whole soybeans, the fibrous hull isremoved and the oil is extracted. The remaining soybean meal is acombination of carbohydrates and approximately 50% protein.

Soybean cultivar 8616350112 can be used to produce meal. After oil isextracted from whole soybeans harvested from soybean cultivar8616350112, the remaining material or “meal” is “toasted” (a misnomerbecause the heat treatment is with moist steam) and ground in a hammermill. Soybean meal is an essential element of the American productionmethod of growing farm animals, such as poultry and swine, on anindustrial scale that began in the 1930s; and more recently theaquaculture of catfish. Ninety-eight percent of the U.S. soybean crop isused for livestock feed. Soybean meal is also used in lower end dogfoods. Soybean meal produced from soybean cultivar 8616350112 can alsobe used to produce soybean protein concentrate and soybean proteinisolate.

In addition to soybean meal, soybean cultivar 8616350112 can be used toproduce soy flour. Soy flour refers to defatted soybeans where specialcare was taken during desolventizing (not toasted) to minimizedenaturation of the protein and to retain a high Nitrogen SolubilityIndex (NSI) in making the flour. Soy flour is the starting material forproduction of soy concentrate and soy protein isolate. Defatted soyflour is obtained from solvent extracted flakes, and contains less than1% oil. Full-fat soy flour is made from unextracted, dehulled beans, andcontains about 18% to 20% oil. Due to its high oil content, aspecialized Alpine Fine Impact Mill must be used for grinding ratherthan the more common hammer mill. Low-fat soy flour is made by addingback some oil to defatted soy flour. The lipid content varies accordingto specifications, usually between 4.5% and 9%. High-fat soy flour canalso be produced by adding back soybean oil to defatted flour at thelevel of 15%. Lecithinated soy flour is made by adding soybean lecithinto defatted, low-fat or high-fat soy flours to increase theirdispersibility and impart emulsifying properties. The lecithin contentvaries up to 15%.

For human consumption, soybean cultivar 8616350112 can be used toproduce edible protein ingredients which offer a healthier, lessexpensive replacement for animal protein in meats, as well as indairy-type products. The soybeans produced by soybean cultivar8616350112 can be processed to produce a texture and appearance similarto many other foods. For example, soybeans are the primary ingredient inmany dairy product substitutes (e.g., soy milk, margarine, soy icecream, soy yogurt, soy cheese, and soy cream cheese) and meatsubstitutes (e.g., veggie burgers). These substitutes are readilyavailable in most supermarkets. Although soy milk does not naturallycontain significant amounts of digestible calcium (the high calciumcontent of soybeans is bound to the insoluble constituents and remainsin the soy pulp), many manufacturers of soy milk sell calcium-enrichedproducts as well. Soy is also used in tempe, where the beans (sometimesmixed with grain) are fermented into a solid cake.

Additionally, soybean cultivar 8616350112 can be used to produce varioustypes of “fillers” in meat and poultry products. Food service, retail,and institutional (primarily school lunch and correctional) facilitiesregularly use such “extended” products, that is, products which containsoy fillers. Extension may result in diminished flavor, but fat andcholesterol are reduced by adding soy fillers to certain products.Vitamin and mineral fortification can be used to make soy productsnutritionally equivalent to animal protein; the protein quality isalready roughly equivalent.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

The present invention, in various embodiments, include components,methods, processes, systems and/or apparatus substantially as depictedand described herein, including various embodiments, subcombinations,and subsets thereof. Those of skill in the art will understand how tomake and use the present invention after understanding the presentdisclosure.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. In theforegoing Detailed Description for example, various features of theinvention are grouped together in one or more embodiments for thepurpose of streamlining the disclosure. This method of disclosure is notto be interpreted as reflecting an intention that the claimed inventionrequires more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Detailed Description, with eachclaim standing on its own as a separate preferred embodiment of theinvention.

Moreover, though the description of the invention has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the invention (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

Deposit Information

A deposit of the Schillinger Genetics, Inc. proprietary soybean cultivar8616350112 disclosed above and recited in the appended claims has beenmade with the National Collections of Industrial, Food and MarineBacteria (NCIMB), 23 St. Machar Drive, Aberdeen, Scotland, AB24 3RY,United Kingdom. The date of deposit was Feb. 4, 2013. The deposit of2,500 seeds was taken from the same deposit maintained by SchillingerGenetics, Inc. since prior to the filing date of this application. Allrestrictions will be removed upon granting of a patent, and the depositis intended to meet all of the requirements of 37 C.F.R. §§1.801-1.809.The NCIMB No. is 42104. The deposit will be maintained in the depositoryfor a period of thirty years, or five years after the last request, orfor the enforceable life of the patent, whichever is longer, and will bereplaced as necessary during the period.

What is claimed is:
 1. A seed of soybean cultivar 8616350112, wherein asample of seed of soybean cultivar 8616350112 is deposited under NCIMBNo.
 42104. 2. A soybean plant, or a part thereof, produced by growingthe seed of claim
 1. 3. The plant part of claim 2, wherein the plantpart comprises at least a first cell of said plant.
 4. A soybean plantregenerated from the plant part of claim wherein the plant has all ofthe physiological and morphological characteristics of soybean cultivar8616350112.
 5. A method for producing a soybean seed, comprisingcrossing two soybean plants and harvesting the resultant soybean seed,wherein at least one soybean plant is the soybean plant of claim
 2. 6. Asoybean seed produced by the method of claim
 5. 7. A soybean plant, or apart thereof, produced by growing the seed of claim
 6. 8. A method ofproducing an herbicide resistant soybean plant, wherein the methodcomprises introducing a gene conferring herbicide resistance into theplant of claim
 2. 9. A herbicide resistant soybean plant produced by themethod of claim 8, wherein the gene confers resistance to a herbicideselected from the group consisting of glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,L-phosphinothricin, cyclohexone, cyclohexanedione, triazine,PPO-herbicides, bromoxynil, and benzonitrile.
 10. A method of producinga pest or insect resistant soybean plant, wherein the method comprisesintroducing a gene conferring pest or insect resistance into the soybeanplant of claim
 2. 11. A pest or insect resistant soybean plant producedby the method of claim
 10. 12. The soybean plant of claim 11, whereinthe gene encodes a Bacillus thuringiensis (Bt) endotoxin.
 13. A methodof producing a disease resistant soybean plant, wherein the methodcomprises introducing a gene which confers disease resistance into thesoybean plant of claim
 2. 14. A disease resistant soybean plant producedby the method of claim
 13. 15. A method of producing a soybean plantwith modified fatty acid metabolism or modified carbohydrate metabolism,wherein the method comprises introducing a gene encoding a proteinchosen from phytase, fructosyltransferase, levansucrase, α-amylase,invertase and starch branching enzyme or encoding an antisense gene ofstearyl-ACP desaturase into the soybean plant of claim
 2. 16. A soybeanplant having modified fatty acid metabolism or modified carbohydratemetabolism produced by the method of claim
 15. 17. A method ofintroducing a desired trait into soybean cultivar 8616350112, whereinthe method comprises: (a) crossing a 8616350112 plant, wherein a sampleof seed is deposited under NCIMB No. 42104, with a plant of anothersoybean cultivar having a desired trait to produce progeny plants,wherein the desired trait is chosen from male sterility, herbicideresistance, insect resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, modified seed yield, modified oil percent,modified protein percent, modified lodging resistance, modifiedshattering, modified iron-deficiency chlorosis and resistance toherbicides, insects, bacterial disease, fungal disease or viral disease;(b) selecting one or more progeny plants that have the desired trait toproduce selected progeny plants; (c) crossing the selected progenyplants with the 8616350112 plant to produce backcross progeny plants;(d) selecting for backcross progeny plants that have the desired traitand all of the physiological and morphological characteristics ofsoybean cultivar 8616350112 listed in Table 1; and (e) repeating steps(c) and (d) three or more times in succession to produce selected fourthor higher backcross progeny plants that comprise the desired trait andall of the physiological and morphological characteristics of soybeancultivar 8616350112 listed in Table
 1. 18. A soybean plant produced bythe method of claim 17 wherein the plant has the desired trait.
 19. Thesoybean plant of claim 18, wherein the desired trait is modified fattyacid metabolism or modified carbohydrate metabolism and said desiredtrait is conferred by a nucleic acid encoding a protein selected fromthe group consisting of phytase, fructosyltransferase, levansucrase,α-amylase, invertase and starch branching enzyme or encoding anantisense gene of stearyl-ACP desaturase.
 20. A method of producing acommodity plant product, comprising obtaining the plant of claim 2, or apart thereof, and producing the commodity plant product from the plantor part thereof, wherein the commodity plant product is selected fromthe group consisting of protein concentrate, protein isolate, soybeanhulls, meal, flour and oil.